Molecular dynamics simulations and biochemical characterization of Pf14-3-3 and PfCDPK1 interaction towards its role in growth of human malaria parasite

Ravi Jain; Pinki Dey; Sakshi Gupta; Soumya Pati; Arnab Bhattacherjee; Manoj Munde; Shailja Singh

doi:10.1042/BCJ20200145

Molecular dynamics simulations and biochemical characterization of Pf14-3-3 and PfCDPK1 interaction towards its role in growth of human malaria parasite

Biochem J. 2020 Jun 26;477(12):2153-2177. doi: 10.1042/BCJ20200145.

Authors

Ravi Jain¹, Pinki Dey², Sakshi Gupta³, Soumya Pati¹, Arnab Bhattacherjee², Manoj Munde³, Shailja Singh^{1

4}

Affiliations

¹ Department of Life Sciences, School of Natural Sciences, Shiv Nadar University, Gautam Buddha Nagar, UP 201314, India.
² School of Computational and Integrative Sciences, Jawaharlal Nehru University, Delhi 110067, India.
³ School of Physical Sciences, Jawaharlal Nehru University, Delhi 110067, India.
⁴ Special Centre for Molecular Medicine, Jawaharlal Nehru University, Delhi 110067, India.

PMID: 32484216
DOI: 10.1042/BCJ20200145

Abstract

Scaffold proteins play pivotal role as modulators of cellular processes by operating as multipurpose conformation clamps. 14-3-3 proteins are gold-standard scaffold modules that recognize phosphoSer/Thr (pS/pT) containing conserved motifs, and confer conformational changes leading to modulation of functional parameters of their target proteins. Modulation in functional activity of kinases has been attributed to their interaction with 14-3-3 proteins. Herein, we have annotated and characterized PF3D7_0818200 as 14-3-3 isoform I in Plasmodium falciparum 3D7, and its interaction with one of the key kinases of the parasite, Calcium-Dependent Protein Kinase 1 (CDPK1) by performing various analytical biochemistry and biophysical assays. Molecular dynamics simulation studies indicated that CDPK1 polypeptide sequence (61KLGpS64) behaves as canonical Mode I-type (RXXpS/pT) consensus 14-3-3 binding motif, mediating the interaction. The 14-3-3I/CDPK1 interaction was validated in vitro with ELISA and SPR, which confirmed that the interaction is phosphorylation dependent, with binding affinity constant of 670 ± 3.6 nM. The interaction of 14-3-3I with CDPK1 was validated with well characterized optimal 14-3-3 recognition motifs: Mode I-type ARSHpSYPA and Mode II-type RLYHpSLPA, by simulation studies and ITC. This interaction was found to marginally enhance CDPK1 functional activity. Furthermore, interaction antagonizing peptidomimetics showed growth inhibitory impact on the parasite indicating crucial physiological role of 14-3-3/CDPK1 interaction. Overall, this study characterizes 14-3-3I as a scaffold protein in the malaria parasite and unveils CDPK1 as its previously unidentified target. This sets a precedent for the rational design of 14-3-3 based PPI inhibitors by utilizing 14-3-3 recognition motif peptides, as a potential antimalarial strategy.

Keywords: plasmodium falciparum; 14-3-3 proteins; peptides; protein–protein interaction.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

14-3-3 Proteins / chemistry
14-3-3 Proteins / metabolism*
Amino Acid Sequence
Animals
Malaria, Falciparum / parasitology*
Molecular Dynamics Simulation*
Phosphorylation
Phylogeny
Plasmodium falciparum / growth & development*
Plasmodium falciparum / isolation & purification
Plasmodium falciparum / metabolism
Protein Conformation
Protein Kinases / chemistry
Protein Kinases / metabolism*
Protozoan Proteins / chemistry
Protozoan Proteins / metabolism*
Sequence Homology

Substances

14-3-3 Proteins
Protozoan Proteins
Protein Kinases
calcium-dependent protein kinase-1, Plasmodium falciparum